Abstract HLA-Gpos Treg-mediated suppression is critically dependent on the secretion of IL-10 but not TGF-β. CD4+ T cells constitutively expressing the immune-tolerogenic HLA-G have been described recently as a new type of nTreg (HLA-Gpos Treg) in humans. HLA-Gpos Treg accumulate at sites of inflammation and are potent suppressors of T cell proliferation in vitro, suggesting their role in immune regulation. We here characterize the mechanism of how CD4+ HLA-Gpos Treg influence autologous HLA-Gneg Tresp function. Using a suppression system free of APC, we demonstrate a T–T cell interaction, resulting in suppression of HLA-Gneg Tresp, which is facilitated by TCR engagement on HLA-Gpos Treg. Suppression is independent of cell–cell contact and is reversible, as the removal of HLA-Gpos Treg from the established coculture restored the proliferative capability of responder cells. Further, HLA-Gpos Treg-mediated suppression critically depends on the secretion of IL-10 but not TGF-β.